Evaluation of the mechanical properties and degradation behavior of chitosan-PVA-graphene oxide nanocomposite scaffolds in vitro

Objectives Chitosan (CTS) has been a popular option for scaffold fabrication because of its biocompatibility, biodegradability, antimicrobial and nonimmunogenic effects. However, it is of limited function, due to its low mechanical strength and its solubility in acidified media. These limitations could be overcome by its blending with PVA and incorporation with bioactive materials to improve its mechanical properties and tissue regeneration capability. Methods Carbon based nanomaterials, such as graphene oxide (GO) incorporated with CTS/PVA blend to improve composite-scaffold stability. GO nanoparticles were chemically prepared and fully characterized. Different concentrations of both CTS and nano-GO were used for the fabrication of CTS/PVA/GO nanocomposite films through the solvent-casting method. The mechanical properties, thermal stability biodegradation, and swelling of the nanocomposite films were evaluated after characterization by XRD, FTIR and SEM, to detect the effect of GO incorporation in the scaffold to select the suitable dental application. Results A better performance was observed in thermal stability, biodegradation, and water resistance after GO addition into CTS/PVA scaffolds. Regarding mechanical properties, groups were assessed by Kruskal Wallis test afterward Dunn’s post hoc test. There was no significant difference in tensile strength between the nanocomposite films of CTS (2%) and CTS (3%). The tensile strength decreased after addition of nano-GO at different concentrations. The elastic modulus significantly increased when (1%) GO was added into the 1CTS (2%):1PVA. Conclusions CTS/PVA/GO nanocomposite can be used in dental hard tissue engineering, as the incorporation of GO into the CTS/PVA polymer blend improves its properties which is regarded as the critical concentrations of CTS and GO.


Abstract
Objectives: Chitosan (CTS) has been a popular option for scaffold fabrication because of its biocompatibility, biodegradability, antimicrobial and nonimmunogenic effects.However, it is of limited function, due to its low mechanical strength and its solubility in acidified media.These limitations could be overcome by its blending with PVA and incorporation with bioactive materials to improve its mechanical properties and tissue regeneration capability.
Methods: Carbon based nanomaterials, such as graphene oxide (GO) incorporated with CTS/PVA blend to improve composite-scaffold stability.GO nanoparticles were chemically prepared and fully characterized.Different concentrations of both CTS and nano-GO were used for the fabrication of CTS/PVA/GO nanocomposite films through the solvent-casting method.The mechanical properties, thermal stability biodegradation, and swelling of the nanocomposite films were evaluated after characterization by XRD, FTIR and SEM, to detect the

Introduction
The human body is a complex structural system with multiple types of cells and tissues.Researchers are studying the applications of multiple biomaterials to repair defects in the body.1e3 Tissue engineering is aimed at regenerating damaged tissues through biologically compatible techniques.The main components of tissue engineering are cells, growth factors, and scaffolds. 1Optimization of scaffolds can create ideal conditions for cellular growth.For effective use, scaffolds must meet certain criteria, including sufficient biocompatibility, porosity, degradation rate, mechanical strength, sterility, and cost-effectiveness. 4oth organic and synthetic polymer-based biomaterials are used for scaffold construction.Natural polymers including collagen, gelatin, chitosan (CTS), cellulose, and alginate, are biocompatible agents that can increase cellular adhesion and proliferation.However, they lack the mechanical strength necessary for some applications, such as hard tissue engineering. 5Polyvinyl alcohol (PVA) is considered a favorable candidate for tissue regeneration, because of its controlled biodegradability, but it has several disadvantages, such as poor migration of metabolites and stimulation of inflammatory reactions during its degradation.In general, degradable polymers have very low mechanical properties, whereas mechanically strong polymers are always bioinert. 6,7S-based biomaterials have received substantial attention and are widely used in a variety of applications, because of their specific characteristics, including low foreign body reactions, antibacterial activity, biodegradability, and biocompatibility. 8CTS can form porous structuresda key characteristic in tissue engineering and cell transplantation.CTS is derived from chitin, which is present in the exoskeletons of crustaceans. 9This linear, pseudo-natural, and semi-crystalline polysaccharide is composed of (1 / 4)-2-acetamido-2-deoxy-b-D-glucan (N-acetyl Dglucosamine) and (1 / 4)-2-amino-2-deoxy-b-D-glucan (Dglucosamine). 10However, chitin is difficult to use in biomedical applications because it is insoluble in water.Consequently, chitin is deacetylated and transformed to CTS.Enzymatic or alkaline deacetylation removes the acetyl group from the C-2 position of chitin, thereby resulting in insertion of a primary amine group.Chitin becomes soluble in acidic media when its acetylation level exceeds 50%, because of protonation of the amine group. 10,11The primary amine group, which has a positive charge and is responsible for CTS's ability to interact with negatively charged macromolecules, such as DNA, RNA, and other biological components, is also responsible for CTS's antibacterial activity, muco-adhesiveness, and hemostatic properties. 12,13CTS, like many polysaccharides, has several inherent disadvantages, such as poor solubility and stability in physiological fluids, and insufficient flexibility; consequently, it is rarely used in its pure form and instead is combined with other materials. 14VA is a synthetic water-soluble polymer with exceptional biocompatibility, nontoxicity, and non-carcinogenicity; thus, it is considered an ideal biomaterial for use in polymer blends. 15,16PVA also can increase the flexibility of other polymers. 16The CTS/PVA polymer blend becomes nonmiscible at high concentrations of PVA (>50%).Miscibility occurs only at lower PVA concentrations, because of PVA's weak intermolecular attraction and strong intramolecular structure. 17CTS/PVA blends are used for multiple medical purposes, e.g., tissue engineering, 18 wound dressings, 19 and drug delivery systems. 20Recently, numerous inorganic substances, including clays and ceramic particles, have been applied as reinforcing agents to modify the final polymer's characteristics and performance. 21Additionally, carbonbased nanomaterials, including carbon nanotubes, graphene, and graphene oxide (GO), are currently used because of their exceptional mechanical, optical, electrical, and biological properties. 22GO nanosheets provide a biocompatible platform for cellular growth and tissue regeneration. 23These materials have also received substantial interest in a variety of biomedical applications, including drug delivery, 24 bioimaging, 25 tissue engineering, 26 and antibacterial treatments. 27GO can be prepared through the modified Hummer method with a high product yield. 28ecause GO has oxygen-containing functional groups, such as epoxy, carboxyl, and hydroxyl groups, on its nanosheet basal plane and edge, it is more hydrophilic and dispersible in water than graphene and graphite powder. 29,30hese functional groups engage in strong interactions with polar solvents and polymer matrices, thus facilitating GO dispersion. 31GO is also effective against Gram-positive and Gram-negative bacteria.32e34 This antibacterial action occurs through various mechanisms, including high oxidative stress, membrane stress and cell wrapping; GO has also been found to promote cell proliferation in a dosedependent manner. 35,36Graphene-based coatings have been reported to protect implants against corrosion by preventing bacterial colonization. 37O positively influences the proliferation and differentiation of mesenchymal stem cells, as reported in some in vitro cultures. 38,39Moreover, GO has the necessary properties for the development of biosensors, including good electrical conductivity, large surface area, and high electromechanical activity. 40Biopolymer-GO applications have been reported, given that GO is considered nontoxic and can improve polymer properties even at very low concentrations, in contrast to other reinforcing fillers. 41erein, we focused on evaluating the mechanical properties, biodegradation behavior, water sorption, and thermal stability of CTS-PVA-GO nanocomposites with different ratios of CTS and GO in the scaffold composition.The null hypothesis was that no difference would be observed in the mechanical properties, biodegradation, water sorption, and thermal stability of different concentrations of CTS and GO in a nanocomposite scaffold.

Study design
In this in vitro study, 136 samples were used for characterization, and the evaluation of mechanical properties, biodegradation, and water sorption.The sample size was calculated on the basis of a 95% confidence level and 80% study power with Rosner's method 42 in G*Power software version 3.0.10(G*Power 2019, Universita¨t Du ¨sseldorf, Germany).The study samples were divided into eight groups according to the tested concentrations of CTS (2% or 3%) and GO (0%, 0.3%, 0.5%, and 1%).Each group comprised 17 samples: five samples for characterization, eight samples for evaluation of mechanical properties, two samples for biodegradation assessment, and two samples for swelling measurement.

Preparation of GO nanoparticles
GO was synthesized through a slightly modified Hummer method, as previously reported. 43Graphite powder (3 g) was added to 70 ml concentrated H 2 SO 4 , and the mixture was stirred with a mechanical stirrer for 10 min while being kept in an ice bath and monitored with a thermometer to regulate the temperature.Over 1 h, 9.0 g KMnO 4 was gradually integrated into the suspension and stirred at room temperature.Subsequently, 5.0 g potassium persulfate was gradually added to the suspension solution.
To prevent a sudden rise in reaction temperature, an ice bath and thermometer were used to maintain a constant temperature of 25 C.After 1 h, the suspension solution was added to 500 ml distilled water, and 15 ml hydrogen peroxide was subsequently added.The resulting solid was filtered and rinsed once with double-distilled water, once with acidic water (10% HCl), and twice with double-distilled water.The resultant substance was dried at 70 C for 48 h, then stored for subsequent experiments. 43aracterization of GO nanoparticles GO was characterized through the following methods: Raman spectroscopy, UV/visible spectrophotometry (double beam model T80þ, PG instruments Ltd., UK), FTIR (BRUKER, Germany) covering the spectrum range of 400e 4000 cm À1 , X-ray diffraction analysis (XRD) (XRD-7000 Shimadzu, Japan), SEM, and EDX (JEOL, JSM-5 investigator model, Japan).

Preparation of CTS/PVA/GO nanocomposite films
Two concentrations of CTS were prepared (2% and 3% [wt./v]) by dissolving CTS in 2% acetic acid under stirring for 5 h at room temperature. 44Subsequently, 10% PVA solution was prepared in distilled water at 60 C with stirring for 2 h.The polymer blends were obtained by mixing two polymeric solutions (50% CTS:50% PVA), as reported elsewhere. 45In a solution containing 2% acetic acid, GO was dispersed at three concentrations (0.3%, 0.5%, and 1% [wt./v]).Sonication was used to ensure uniform distribution of the mixture. 44,45CTS/PVA/GO nanocomposite films were obtained through the solvent-casting method.GO solution was added to the polymer blend and stirred for 24 h until a homogeneous mixture was obtained.The solution was transferred into a Petri dish, and the casted film was dried in a vacuum oven at 60 C overnight. 44

Instrumental characterization of prepared CTS/PVA/GO nanocomposite films
The thermal behavior of the nanocomposites was evaluated through thermal gravimetric analysis (TGA).Thermal mechanical and degradation of chitosan-PVA-GO stability was evaluated with TGA/DCS (Shimadzu, Japan) under a nitrogen atmosphere.Evaluation of the mechanical properties of the nanocomposite was conducted through two modes as follows.(a) Tensile strength tests were conducted according to ISO 2062:2009 specifications.The extension rate was kept at 5 mm/min, and the cell load was fixed at 20 N with a gauge length of 30 mm. 44 The dimensions of the samples were 6 cm Â 1 cm. 46The tensile strength was evaluated for both CTS/PVA and CTS/PVA/GO nanocomposite films (Fig. S1, supplementary data).(b) In Young's modulus evaluation, the elastic modulus in both CTS/PVA and CTS/PVA/GO nanocomposite films was evaluated to assess the effect of GO incorporation on the scaffold.

In vitro biodegradation assessment
Samples of CTS/PVA and CTS/PVA/GO nanocomposite films (10 Â 10 mm) were placed in 5 ml PBS (pH ¼ 7.4) to evaluate the biodegradation rate.The samples were weighed and then stored in an incubator at 37 C.After 7, 14, 21, and 28 days, samples were removed from the PBS, rinsed in double-distilled water, dried in a 40 C oven, and weighed again.Final calculations of the weight loss percentage (WL) were performed according to the following equation: where W0 is the weight of the sample before soaking, and W1 is the weight of the sample after soaking. 44elling study Gravimetric measurements were conducted to assess the films' swelling kinetics under ambient conditions.The films were fully dried in a vacuum oven, and their weight (Wi) was determined; they were then placed in a beaker containing 50 ml saline.After removal of surface water with a filter paper, the difference in the film's weight (Wt.) was measured after 1, 3, 24, and 48 h with the following equation 14 : Swelling ratio (%) ¼ [(Wt.-Wi)/Wi]Â 100. (2)

Statistical analysis
Variables are presented as median, interquartile range (IQR), minimum, and maximum values, in addition to mean and standard deviation (SD).Variables were compared with the Kruskal Wallis test followed by Dunn's post hoc test with Bonferroni correction.The significance level was set at a P value of 0.05.Data were analyzed in IBM SPSS (version 23.0).Abbreviations CTS, Chitosan; PVA, Polyvinyl alcohol; and GO, Graphene oxide.

Raman spectroscopy
In the Raman spectra of GO (Figure 1), the G band was broad, and shifted to n 1605 cm À1 .The D band had high intensity because of disordering in the sp 2 structure and was observed at n 1354 cm À1 .

FTIR analysis
The spectrum of GO (Figure 2a) exhibited multiple characteristic peaks corresponding to multiple oxygencontaining functional groups, thus confirming the formation of GO nanosheets.Bands at approximately n 1709 cm À1 , n 1091e1042 cm À1 , and n 3243 cm À1 corresponded to C¼O (carboxyl or carbonyl), CeO (epoxy or alkoxy), and OeH stretching of the COOH group, respectively.The peak around n 811 cm À1 was attributed to aromatic CeH deformation.Spikes at n 646, 575, and 495 cm À1 were attributed to CeH bending vibration.The band at n 1627 cm À1 was associated with the remaining sp 2 structure.

XRD analysis
The spectrum of the prepared GO (Figure 3a) exhibited a characteristic diffraction pattern at 2q of 8 , which corresponded to an interlayer distance of the (001) plane of approximately 12.4 A ˚. Another diffraction peak observed at 26.9 corresponded to the (002) plane and d spacing of 3.4 A ˚.This peak appeared because of the remaining graphitic sp 2 structure.

SEM investigation
As shown in Figure 4a, the synthesized GO had a layered structure associated with ultrathin homogeneous films that were either folded or continuous.The sheets' edges showed kinked or wrinkled areas.GO particles were sometimes associated and formed aggregates.

EDX analysis
According to the elemental analysis of GO mass (Fig. S2, supplementary data), an increase in the content of oxygen atoms was observed, because GO is an oxygen rich material with a composition of 55 at% carbon and 44 at% oxygen.The C/O ratio was approximately 1.25, thereby suggesting that a high degree of oxidation by the strong oxidant (KMNO 4 ) led to shifts in the distances of GO layers.

Characterization of prepared CTS/PVA/GO nanocomposite films Macroscopic investigation of CTS/PVA/GO nanocomposite films
In general, CTS/PVA films are yellowish and somewhat translucent because of the PVA.The addition of GO made the films turn black.The intensity of the black color increased with increasing GO amount (Fig. S3, supplementary data).The film thickness, measured with digital calipers, was found to be 0.2 mm.

XRD analysis
XRD patterns of CTS/PVA and CTS/PVA/GO at different concentrations are shown in Figure 3b.XRD of the CTS/PVA polymer blend showed a sharp diffraction peak at a 2q of approximately 21 , which was associated with crystallites of PVA.With the addition of GO, the nanocomposite showed identical crystallinity to that of the polymer blend, and the same diffraction peak; this peak showed a slight shift to a lower value when the GO amount was increased.Another absorption peak observed at n 1086 cm À1 corresponded to CeO stretching.The CTS spectrum (Figure 2c) showed a broad band at 3360 cm À1 , because of the overlapping of eOH and eNH stretching vibrations.The peak at 2868 cm À1 corresponded to the stretching vibration of CH 2 .Another band at n 1590 corresponded to eNH bending (NH 2 ).Carbonyl stretching from CeH bending and CeOeC linking were associated with the bands at n 1376 and 1150 cm À1 .In the CTS/PVA polymer blend (Figure 2d), the intensity of the n 3279 cm À1 peak decreased to n 3268 cm À1 .Additionally, substantial intensity of the absorption band corresponding to CeH stretching vibration was observed at n 2938 cm À1 .The peak at n 1327 cm À1 also confirmed the crosslinked structure with PVA, because of the deformation vibration of CH 2 .The peak at n 1651 cm À1 corresponded to C¼N stretching, owing to the reaction of the NH 2 groups of CTS with eOH groups associated with PVA.
In CTS/PVA/GO nanocomposites (Figure 2e), the strong characteristic peak at n 3268 cm À1 was associated with the e OH group present in GO and the stretching of the NH 2 group in CTS.The peaks at n 1410 and 1143 cm À1 were associated with carboxylate vibrations.The vibrational peak at n 1643 cm À1 was associated with the deformation vibration of absorbed H 2 O molecules.Another characteristic peak at n 1554 cm À1 was associated with the NH 2 groups of CTS.The peak at n 2907 cm À1 was associated with CeH stretching vibration.The bands at n 923 and 847 cm À1 were associated with the polysaccharide structure present in CTS.

SEM investigation
The microstructures of the CTS/PVA polymer blend and nanocomposite films were assessed by SEM (Figure 4).The polymer blend (Figure 4b) showed a smooth surface indicating uniform dispersion and good miscibility between polymers.The surfaces of the pure CTS and pure PVA films were relatively smooth; consequently, the blend was expected to show the same surface morphology.The morphology of the nanocomposite (Figs.4c, 4d and 4e) showed a rough surface associated with GO dispersion.As the ratio of GO increased, the entire polymer surface was coated by GO, and the surface became rougher and less visible, owing to the substantial interface between GO and the polymer.

TGA
TGA is a valuable method for evaluating polymer stability.As shown in Figure 5, the addition of GO to the polymer blend shifted the degradation to a higher temperature.For the polymer blend (1CTS:1PVA) (Figure 5a), the first degradation step was detected between 29 C and 125 C, and a 14% weight loss was associated with breakdown of the polymer backbone.Lower weight loss percentages of 10.7% and 11% in the first degradation step were observed with 0.3% GO (Figure 5b) and 0.5% GO (Figure 5c), respectively.For the polymer blend (1CTS:1PVA) (Figure 5a), the major decomposition temperature ranged from 260 C to 396 C, and the weight loss was approximately 30%.Major weight loss occurred at higher temperatures (323e499 C) with the addition of 0.3e0.5% GO (Figures 5b and 5c, respectively).When the GO loading was increased to 1% (Figure 5d), the major degradation temperature was lower (260e378 C).An increase in the first degradation step, to 16.5%, was observed with 1% compared with 0.3e0.5% GO.The residue mass was 20% for 1CTS:1PVA, and 26%, 23%, and 21% for 0.3%, 0.5%, and 1% GO dispersions, respectively.

Discussion
The production of GO depends on multiple factors including the acid concentration, strength of the oxidizing agent, and decay of the formed intermediate compound.Addition of strong sulfuric acid to graphite leads to intercalation and formation of graphite bisulfate (a graphite intercalated compound).After expansion of the graphene layer, another intercalation occurs at the basal plane. 47orphization occurs gradually, and the interlayer distance increases.Additionally, the lattice parameter along the caxis and the number of layers decrease.With addition of KMNO 4 , ultrasonic cleavage of graphite oxide with substantial numbers of surface eOH, eCOOH, and epoxy groups occurs.These oxygen functional groups increase the distance between layers and make the structure more hydrophilic. 47,48aman spectroscopy is used to verify the disorder and weaknesses in the crystal structures of carbonaceous materials.The G band arises from CeC bond stretching.As the disorder of graphite increases, this G band becomes broad at n 1605 cm À1 , and a broader D band is observed at n 1354 cm À1 .According to Yuan et al. (2017), the presence of broad and intense G and D bands is associated with higher degrees of oxidation than observed in other samples with lower oxidation. 49Additionally, the I D/ I G ratio describes the sp 2/ sp 3 carbon ratio, which is associated with the number of change sides made by the functional groups' attachment to carbon, an indicator of the aromatic structure's integrity.A ratio reaching 0.8 indicates structural defects produced by oxidation and attachment of the functional groups. 50,51TIR characterization of GO revealed the presence of oxygen atoms in the form of eOH, C¼O, and CeO, thereby confirming the oxidation process and GO formation.These hydrophilic functional groups are highly important in the compatibility between GO and the polymeric matrix.Additionally, these abundant functional groups have been found to improve GO hydrophilicity. 46In the oxidation process of graphite by KMNO 4 , an Ogroup was added to the structure, thus potentially increasing the distance between layers from 3.4 to 12.4 A ˚, because of the repulsion forces between layers.According to Absazade, 52 the oxidation process of graphite leads to narrowing of the graphitic characteristic peak at 2q ¼ 26.9 and the formation of a new peak at 2q ¼ 8 which is characteristic of GO.These changes are due to the heterogeneous nature of the oxidized graphite, which contains domains of graphite (sp 2 ) and oxidized graphite (sp 3 ).The oxidation process was also confirmed through elemental analysis by EDX, which yielded results in line with those of Siburian et al. 53 For preparation of the nanocomposite, CTS was added to PVA, and various GO concentrations (0.3%, 0.5%, and 1%) were added to the polymer blend.The addition of CTS to PVA decreased its crystallinity; consequently, the characteristic peak of PVA in the XRD pattern shifted to a lower value.On the basis of findings from Saeedi et al., 54 the CTS characteristic peak was expected to appear at 2q ¼ w20.3 ; however, it was not detected, to be confirming the good compatibility between polymers.Additionally, the characteristic peak of GO was not observed, owing to the good dispersion of nanoparticles and their presence in trace amounts.
FTIR analysis of pure PVA revealed a spectrum associated with its hydroxyl groups.The combination of frequencies was produced by the stretching vibrations of the backbone aliphatic CH, CHeOH, and CO.The CTS spectrum showed bands associated with NeH and CeH stretching.For the CTS/PVA polymer blend, the increase in the intensity of CeH stretching indicated good miscibility between PVA and CTS.The lower intensity of the band associated with eOH stretching in the polymer blend than observed for pure PVA might have been due to the vibrational stretching of the eOH of PVA with secondary eNH groups associated with CTS through intermolecular hydrogen bonding.The presence of GO in the blend was indicated by the formation of a hydrogen bond between GO and the polymer.The band at n 3268 cm À1 appeared to increase to n 3271 cm À1 , because of the stretching vibration of eOH groups associated with GO. 55 GO addition to the CTS/PVA blend increased the thermal stability, because the GO provided a physical barrier that caused scaffolds to decompose more slowly with increasing temperature.Furthermore, hydrophilic and electrostatic interactions formed between CTS and GO, and hydrogen interactions formed between the polymer chains and GO, thus limiting the motion of polymer chains, in addition the good dispersion of GO within the polymer blend.As the GO dispersion ratio increased to 1%, the major degradation temperature decreased, and the residue weight was lower (21%) than that with 0.3% GO (26%) and 0.5% GO (23%).These findings were probably due to the aggregation of GO nanoparticles inside the polymer matrix. 56,57he tensile strength of the films decreased with increasing CTS concentration, possibly because excessive CTS prevented the GO sheets from assembling into high-orientation structures; consequently, a substantial number of nanoscale defects limited the materials' performance.In a previous study, a CTS concentration of 0.3% increased tensile strength after addition of GO, but when the CTS concentration was increased to 0.5% and 1%, the tensile strength sharply decreased. 58Because electrostatic interactions are believed to be more powerful than hydrogen bonding, they were considered the main reason for this.Owing to the strong electrostatic interaction between CTS and GO, the addition of excess CTS prevented the formation of a randomly and sparsely distributed structure, and led to precipitation of GO.The orientation of the nanoparticles and their dispersion in the polymeric matrix in a uniform pattern was prevented, thus leading to their aggregation, as previously reported. 59An increase in the elastic modulus of 1CTS (2%):1PVA was observed with addition of 1% GO, because GO comprises a two-dimensional sheet of carbon atoms with covalent bonds and different oxygen functional groups, such as hydroxyl, epoxy, and carbonyl groups on the edges and basal planes.Consequently, numerous intermolecular HeH bonds between GO and CTS can form, thereby improving the elastic modulus of the film. 60This improvement enables application of the scaffold in hard tissue engineering.The addition of 1% GO in 1CTS (3%):1PVA decreased the elastic modulus of the film, a finding potentially associated with increased heterogenicity of the structure with increasing concentrations of both CTS (3%) and GO (1%).Consequently, aggregation of the nanoparticles rather than uniform dispersion would limit use of the scaffold with high GO concentrations in stress bearing areas, as explained by Mombini et al. in a study using carbon nanotubes to improve the mechanical of CTS/ PVA fibers. 61CTS degradation occurs through hydrolysis, wherein water molecules interact with polymeric chains and break them into shorter chains.In the body, lysozyme degrades 1e4N-acetyl glucosamines.Amino sugar is released and subsequently removed from the body via the metabolic system.Saccharides are another byproduct of CTS breakdown that travel through metabolic pathways.Incorporation of GO into CTS and PVA blends significantly increased the stability of the films in PBS, and led to lower weight loss and degradation rates with increasing GO concentrations.These findings might have been due to interactions between COOH groups in GO with CTS leading to an increase in crystallinity and decreasing the degradation rate. 62The HeH bonding between CTS and PVA explains the stability of the polymer blend, given that the film containing CTS completely degraded only after 7 days.
Finally, the water uptake capacity of the nanocomposite films decreased after GO addition to the polymer matrix.GO addition may prevent water diffusion into the film by decreasing the number of hydrophilic groups such as e COOH and eOH, because hydrogen bonding with the eNH of CTS increases the nanocomposite films' water resistance. 63Moreover, the films' rapid water sorption in the first hour enables them to be used as wound dressings with rapid exudate absorption capacity to avoid exudate accumulation.An equilibrium reached after 3 h may maintain the fluid balance necessary for cellular migration.

Limitations
The study did not perform biocompatibility testing or investigate the antibacterial action of the prepared scaffold.

Figure 7 :
Figure 7: Swelling assessment of CTS/PVA/GO nanocomposite films with different concentrations of GO; CTS/PVA blend with 0% GO, blend with 0.3% GO, blend with 0.5% GO, and blend with 1% GO.

Table 2 :
Elastic modulus results for the studied groups of CTS-PVA-GO nanocomposite films.
a Statistically significant difference at P value 0.05.